The present invention relates to the cloning and expression of a gene encoding an enzyme. More particularly, it relates to the cloning and expression of a gene encoding an alcohol dehydrogenase.
Alcohol dehydrogenases (Adhs) (EC 1.1.1.1 NADH! or EC 1.1.1.2 NADPH!) are well studied as a structurally conserved class of enzyme 1!. The X-ray structure of the horse liver primary-alcohol dehydrogenase (1.degree. Adh) is known, and the properties of this enzyme have been extensively detailed 1,2!. Adhs are typically dimeric or tetrameric pyridine dinucleotide-dependent metalloenzymes with a zinc atom involved in catalysis. Adh proteins have been classified as 1.degree. or 2.degree. Adhs based on their relative activities toward 1.degree. and 2.degree. alcohols. It generally has been assumed that 1.degree. and 2.degree. Adhs are structurally similar, and that their substrate differences are due to relatively small changes in their active site architecture. Tetrameric 2.degree. Adhs have been reported from a number of microorganisms 3-7!. The Thermoanaerobacter ethanolicus 39E 2.degree. Adh is a bifunctional Adh/acetylCoA reductive thioesterase 7!. It has been proposed to function physiologically by oxidizing nicotinamide cofactor during ethanol formation, indirectly preventing glycolytic inhibition at the glyceraldehyde dehydrogenase step 8!.
2.degree. Adhs are attractive subjects for use as catalysts of chiral chemical production because of their broad specificities and their highly enantiospecific conversion of prochiral ketones to alcohols 9-12!. Two issues facing the commercial scale application of 2.degree. Adhs in chiral syntheses are the difficulty of regenerating and retaining expensive nicotinamide cofactor and the lack of inexpensive, highly stable enzymes. Cofactor regeneration and retention have been overcome by numerous strategies 13,14!. While thermophilic enzymes are generally much more stable than their mesophilic counterparts, the organisms that produce thermophilic 2.degree. Adhs grow slowly and to low cell densities, making an alternative expression system for these enzymes crucial for both their commercial application and detailed protein structure-function studies.
The discovery of thermophilic bacteria has provided the opportunity to isolate thermostable enzymes directly. The thermophilic anaerobes T. ethanolicus and Thermoanaerobacter brockii 15! express extremely enantiospecific 2.degree. Adhs that are stable above 70.degree. C. These two 2.degree. Adhs have been proposed to be extremely structurally similar based on similar molecular weight and kinetic characteristics 16!. While the Clostridium beijerinckii gene (adhB) encoding a mesophilic NADP(H)-dependent 2.degree. Adh has been cloned and sequenced (Genbank, Acc. no. M84723), and while the amino acid sequence of the T. brockii 2.degree. Adh has been determined by Edman degradation 17!, no cloned thermophilic 2.degree. Adh is available for detailed biochemical studies. The amino acid sequence of the T. ethanolicus 39E 2.degree. Adh was not publically available before the priority date of this patent application.